By age 40 in the US, approximately 55% of the population has been infected with the ?-herpesvirus, HSV1, and between 50-80% of the population has been infected with -herpesvirus cytomegalovirus (CMV). In an immunocompetent host, these infections are controlled by the immune system; however, infections are much more serious in immunocompromised individuals. DNA polymerase inhibitors, acyclovir and valacyclovir, are the mainstay of current anti-HSV therapies and are generally effective in immunocompetent individuals; however, there has been a sharp increase in the onset of HSV resistance in immunocompromised patients. Infections caused by CMV are even more difficult to treat due to resistance and toxicity of the first line therapy, ganciclovir. Immunosuppression leads to reactivation of all herpesviruses and increased levels of resistant virus. Drug resistant virus is more likely to lead to disseminated life-threatening infections such as such as hepatitis, pneumonia, encephalitis, retinitis, and neuropathy. There is a compelling need for the development of safer and more efficacious agents with novel mechanisms of action. All herpesviruses encode a highly conserved alkaline nuclease with DNA exo- and endonuclease activities, and we have shown that the exonuclease activity of the HSV ortholog UL12 is essential for virus growth, suggesting that it may be an excellent target for drug discovery. The viral alkaline nucleases are members of a large superfamily of enzymes known as nucleotidyltransferases (NTS) involved in various aspects of nucleic acid metabolism including DNA replication, repair and recombination. NTS active sites contain a conserved catalytic triad of acidic residues that coordinate strongly to two Mg2+ ions. This configuration can be exploited for the development of small molecule inhibitors. Indeed, we are very encouraged by the successful development of inhibitors of two other viral NTS proteins, HIV integrase and influenza endoribonuclease. We have initiated a program to design small molecules that target the UL12 and UL98 alkaline nucleases. Considerable sequence identity exists between these two orthologs, and we have previously shown that UL98 can functionally complement a UL12 null mutant. We hypothesize that the active site conservation between the alkaline nucleases from HSV and CMV will make it possible to design lead compounds that potently and simultaneously target both herpesvirus nucleases. In support of this hypothesis, we have recently identified three metal-directed pharmacophores that inhibit the enzymatic activities of the alkaline nucleases from HSV (UL12) and CMV (UL98) and exhibit antiviral activity against both viruses in cell-based assays. In this proposal we will perform experiments designed to validate that the alkaline nuclease is the target for the observed antiviral activity, explore residues in the nuclease active sites to improve potency of compounds, and expand our lead series to improve potency and selectivity.